We have solved the crystal structure of the small GTP-binding protein Cdc42 in complex with a C-terminal domain of its GTPase-activating protein (GAP) by a combination of MAD pahsing and a molecular replacement solution. The final model is now refined to 2.1 A resolution to an Rfree of 25.9% and shows the presence of an AlF3 molecule in the active site. The structure of the complex shows the GAP to bind essentially to the switch I and II loops in Cdc42 as well as the end of helix H3. In doing so, the GAP stabilizes one particular conformation of Gln61, a residue that has been found to be essential to all G-proteins. At the same time, the GAP introduces a conserved arginine residue (R305) into the active site to stabilize the negative charge formation on the GTP. The side chain amide groups of R305 are in close contact with one fluorine atom and with the beta-gamma-phosphate-bridging-oxygen. This observation shows that the negative charge is localized on the gamma-phosphate and on the leaving group (the GDP). As a consequence, the transition state has a mixed associative and dissociative structure. To further study the role of R305, we solved the structure of the same complex but with a GAP(R305A) mutant. To our surprise an AlF3 molecule was also found in the active site. The interface between the G-protein and the GAP has not changed. Tyr32 from Cdc42, which was stabilizing R305 in the wild type mutant, has changed its conformation and is now tightly bound to the AlF3. Gln61 in the mutant complex, is seen to be more flexible and to have weaker interactions with the nucleophilic attacking water. Taken together, these results show that GAP is a dual molecule which function is to stabilize the switch domains of the G-protein and to introduce an arginine residue to stabilize the transition formation.